Embryonic cells develop into specialized cell types by a two-step process of determination and differentiation. Determination results in the conversion of embryonic cells into lineages of stem cells which proliferate and eventually differentiate, activating genes that encode the proteins functionally appropriate to each differentiated cell type. Although many experimental systems have described these processes, little is known of the molecular and genetic regulatory mechanisms that mediate conversion of embryonic cells into stem cells and differentiated cell types. To address these questions, we are utilizing the mouse embryonic cell line C3H10T1/2 (10T1/2). These multipotential cells can be converted into stable mesodermal cell lineages representing skeletal muscle, cartilage and fat tissues. We have taken advantage of this in vivo model system by isolating individual cell lines derived from each of these lineages and have shown that the stem cells can be kept in proliferative state indefinitely, or can be induced to differentiate into their respective cell types. In this proposal, our goal is to identify, isolate and characterize the genes and gene products that are required to maintain the multipotential embryonic cell as well as those genes that help define the myogenic and adipogenic cell lineages. Toward this goal, we will construct cDNA libraries that represent mRNA species produced by 10T1/2 cells, myoblasts and preadipocytes. Each of the cDNA libraries will be differentially screened to isolate clones that are expressed exclusively in the multipotential cell state, in determined muscle stem cells or in determined fat stem cells. Each clone will be characterized by examining gene expression patterns in the developing 10T1/2 cell system, in embryonic and adult mice and in mutant 10T1/2 cell lines that are determination and/or differentiation defective. In addition, expression vectors containing unique cDNA clones will be used to generate and/or rescue other developmental mutants. These studies will help determine how individual genes interact to establish and maintain each of the three cell lineages and should provide important information regarding the molecular processes involved in embryonic development.